Energy status regulates levels of the RAR/RXR ligand 9-cis-retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas β-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the β-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.

The parent retinoid, retinol, produces multiple metabolites, with all-trans-retinoic acid (atRA) functioning as a major autacoid (1,2).atRA activates retinoic acid (RA) receptors (RAR), but also acts through noncanonical mechanisms (3,4).An atRA isomer, 9-cis-retinoic acid (9cRA) binds with high affinity to both RAR and retinoid X receptors (RXR) in vitro (5).9cRA occurring in tissue concentrations similar to atRA would verify presence of an autacoid with potentially more extensive regulatory impact than atRA.Since its identification as a high-affinity RAR and RXR ligand, however, the in vivo occurrence of 9cRA has been controversial (5)(6)(7).Multiple searches failed to demonstrate 9cRA as an endogenous tissue retinoid (8).These included liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays validated for tissue analyses of RA isomers (9)(10)(11).Inability to quantify 9cRA in vivo initiated searches for an endogenous RXR ligand.Phytanic acid, docosahexaenoic acid, and several polyunsaturated fatty acids were proposed as RXR ligands, but each requires 4 to >10 μM to activate RXR, that is, several orders of magnitude less potent than 9cRA (12)(13)(14)(15).None attains in vivo concentrations as free acids capable of activating RXR, and none activate RAR.A more recent conclusion that 9-cis-13,14dihydroretinoic acid serves as a physiological RXR ligand faces several drawbacks (16).It does not bind RAR at all, and its more active R-enantiomer has no affinity for RXR at 100 nM; at 10 μM it exerts only 40% of 9cRA's binding affinity.Its affinity was estimated by a reporter assay with an RXR ligandbinding domain transfected into COS cells, which did not achieve saturation.These limitations pose substantial concern about the relevance of 9-cis-13,14-dihydroretinoic acid as an RXR ligand, especially if 9cRA were present in tissues.
The question of 9cRA as an endogenous retinoid was partially resolved by its quantitation in pancreas in concentrations similar to or higher than atRA (17,18).Pancreas 9cRA relates inversely to serum glucose and insulin in mice, and induces glucose intolerance during glucose-tolerance tests.9cRA in nanomolar concentrations represses glucosestimulated insulin secretion (GSIS) in pancreatic β-cells and in mouse islets through inhibiting glucose uptake by Glut2 and glucose phosphorylation by glucokinase (Gck), posttranscriptionally.9cRA also suppresses Pdx-1 and HNF4α mRNA transcriptionally.Pdx-1 induces Gck, whereas HNF4α induces Glut2, and both stimulate insulin release through enhancing mitochondrial metabolism (19)(20)(21).Defects in GK, Pdx-1, and HNF4 underlie maturity onset diabetes of the young (MODY1, 2, and 4).9cRA decreases in mouse models of reduced β-cell content, such as heterozygous Akita mice and streptozotocintreated mice.In contrast, 9cRA rises to abnormally high levels in glucose-intolerant mice with β-cell hypertrophy, including mice with diet-induced obesity, as well as ob/ob and db/db mice.These data established 9cRA as a pancreatic autacoid that modifies GSIS, and beg the question whether 9cRA occurs endogenously in other tissues.
Widespread occurrence of 9cRA in vivo would inform its therapeutic applications.As the drug alitretinoin, 9cRA exerts ameliorating effects on skin cancers, such as cutaneous T-cell lymphoma and Kaposi's sarcoma, therapy-resistant lichen planus, and chronic hand eczema (22)(23)(24).9cRA also has been evaluated for chemoprevention and chemotherapy, because it promotes neuroblastoma cell differentiation, inhibits growth of the human myeloid leukemia cell line HL-60 with greater potency than atRA, induces differentiation of isolated human leukemia cells, and inhibits growth of the estrogen receptorpositive breast cancer cell line MCF7 (25,26).
Potential for new therapeutic agents has spawned synthesis and evaluation of 9cRA analogs that bind specifically with RXR, known as rexinoids (27,28).The Food and Drug Administration has approved one of these, Targretin (bexarotene), for treating cutaneous T cell lymphoma (29,30).Rexinoids also decrease hyperglycemia and hyperinsulinemia by improving skeletal muscle insulin sensitivity in mouse models of obesity and noninsulin-dependent diabetes (31)(32)(33)(34)(35).Although rexinoids may prove useful as insulin sensitizers and to reduce obesity, they also present with deleterious side effects.These include suppressing thyroid hormone action, increasing serum triglycerides and inducing hepatomegaly (34).The diverse actions of rexinoids likely result from RXR heterodimerization with type II nuclear receptors, including FXR, LXR, PPAR, PXR, RAR, THR, and VDR (36).Understanding locations and regulation of 9cRA in tissues should provide data useful to evaluating clinical application of rexinoids.
The present work aimed to determine whether 9cRA occurs in tissues other than pancreas, and if so, to provide insight into its presence vis-à-vis atRA, and regulation of its biosynthesis.We report widespread tissue occurrence of 9cRA in concentrations similar to atRA.9cRA and atRA vary tissue specifically and independently in tissues with fasting versus refeeding.In β-cells, glucose reduces Rdh5-catalyzed 9cRA biosynthesis, accounting for decreased 9cRA during the fed state.Variation of 9cRA with energy status complements actions of type II nuclear receptors, which regulate genes that modify energy metabolism, among other functions.

Prevention of RA isomerization
Skepticism about 9cRA as an autacoid has been fostered by failure to identify it in tissues using analyses optimized for quantifying atRA.To address this, we revised recovery and liquid chromatography (LC) protocols for quantifying RA isomers in serum and tissues by returning to tissue homogenization with methanol, rather than with saline/ethanol, and modifying LC elution to improve resolution (37).The original LC mobile phase resolved four RA isomers (Fig. 1).The revised LC protocol improved resolution (Fig. 1, B and C).The original saline/ethanol homogenization resulted in atRA isomerizing partially into 13cRA (Fig. 1D) and 9cRA isomerizing partially into atRA (9,13-di-cis-RA (9,13dcRA) and 13cis-RA (13cRA) (Fig. 1F).In contrast, methanol-based 9-cis-retinoic acid occurs as an energy-regulated autacoid homogenization maintained the integrity of extracted atRA and 9cRA (Fig. 1, E and G).To verify reliability of the methanol-based procedure, we added 9cRA to a pancreas homogenate, extracted the sample, and compared it to a sample without added 9cRA (Fig. 2).The methanol-based procedure recovered 9cRA without isomerization.

Full-scan mass spectrum of endogenous 9cRA
To further confirm 9cRA authenticity, 46 pooled mouse pancreata were analyzed after methanol homogenization, followed by extraction with hexane.Endogenous 9cRA was resolved from other RA isomers by high-performance LC.Reinjection of the collected 9cRA fraction for a Q3 mass scan generated a mass spectrum consistent with that of a 9cRA standard (Fig. 3) (38).The protonated molecular ion occurred at m/z 301.Characteristic product ions were present at m/z 283, 255, 205, and 123.These ions identified the molecular weight as an RA, with loss of water, loss of the carbonyl group, presence of the side-chain plus a portion of the β-ionone ring and the β-ionone ring, respectively.

Assay of tissue RA isomers
Both protocols were applied to mouse tissues to determine impact on the composition of RA isomers.Identification of RA isomers consisted of LC comigration with authentic standards, selection of the molecular ion at m/z 301 by Q1, and quantification of the characteristic product ion at m/z 205 in Q3.The ethanol protocol provided results equivalent to the original reports authenticating 9cRA in pancreas and presence of  9-cis-retinoic acid occurs as an energy-regulated autacoid 9,13dcRA, but not 13cRA (Fig. 5A), and failing to observe 9cRA above the limits of quantification outside pancreas (17,18).Serum and liver included atRA, relatively high concentrations of 9,13dcRA, and undetectable 13cRA, as reported originally.The brain showed a high level of 9,13dcRA, with atRA and 9cRA below limits of quantification.A screen of kidney, testis, epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) and brown adipose tissue (BAT) revealed similar results.Comparatively, the methanol-based protocol revealed presence of 9cRA and atRA in the pancreas, but did not include 9,13dcRA or 13cRA (Fig. 5B).The methanol-based protocol showed 9,13dcRA only in serum, confirming a previous report (39).The revised protocol detected 9cRA in all other tissues assayed: the liver, the brain, the kidney, testes, eWAT, BAT, and iWAT, and did not show 9,13dcRA.
Fasting affects 9cRA and atRA tissue concentrations independently 9cRA concentrations exceeded atRA in the brain, the kidney, eWAT and fasted pancreas (Table 1).atRA concentrations exceeded 9cRA in the liver and testis.9cRA and atRA occurred at comparable levels in BAT and iWAT.A 16-h fast increased 9cRA 3-fold and atRA 2-fold in pancreas and >4-fold in eWAT.9cRA levels also were increased by fasting in the BAT (1.7-fold) and brain (2.3-fold), whereas atRA levels were not affected.Fasting decreased 9cRA 50% in the kidney.Fasting increased atRA 1.9-fold in the liver, consistent with previous reports (40)(41)(42).Fasting/refeeding did not affect atRA levels in serum, BAT, iWAT, kidney, brain, and testis.

Glucose suppresses Rdh5 transcription
Tissues express Rdh5 widely and recombinant Rdh5 catalyzes conversion of 9-cis-retinol into 9cRA efficiently (46).Decreases in 9-cis-retinal and 9cRA with 15 mM glucose suggest that refeeding versus fasting affects Rdh5 mRNA or Rdh5 activity, as it does for Rdh1 and Rdh10 in the liver (40)(41)(42).Refeeding after a fast decreased Rdh5 mRNA in pancreas, with reductions of 40 and 67% in 4 and 6 h, respectively (Fig. 7A).Reductions after refeeding were not limited to pancreas (Fig. 7B).Six hours after refeeding Rdh5 mRNA decreased in the liver, kidney, and brown adipose tissue by 68, 93, and 92%, respectively, but increased 15% in eWAT and 80% in retinal pigment epithelium.Regulation of Rdh5 mRNA by glucose was tested in 832/13 cells (Fig. 7C).Glucose (15 mM) reduced Rdh5 mRNA 60% relative to 3 mM glucose after 6 h, without affecting Rdh10.We also evaluated the 9-cis-retinoic acid occurs as an energy-regulated autacoid mouse pancreas cell line MIN6, another model of β-cell function (Fig. 7D) (47).Rdh5 mRNA in the presence of 3 mM glucose exceeded that of 15 mM glucose by 1.8-fold.

Glucose reduces 9cRA biosynthesis through Akt independent of insulin
We tested insulin to determine the possibility of negative feedback on 9cRA biosynthesis (Fig. 8A).Insulin had no effect on Rdh5 mRNA, regardless of the medium glucose concentration.We then applied pharmacological approaches to major aspects of glucose signaling to identify pathways linking glucose to Rdh5 expression.Glucose increases cAMP in β-cells and 3-isobutyl-l-methylxanthine (IBMX) increases cAMP and cGMP (48).IBMX decreased Rdh5 expression by 52 to 56% in 3 mM and 15 mM glucose, respectively (Fig. 8B).Glucagonlike peptide-1 (GLP-1) targets β-cells, among others, and increases β-cell cAMP (49).The GLP-1 analog exendin-4, however, had no effect on Rdh5 mRNA, regardless of the glucose concentration.Increased glucose via cAMP activates Crem (cAMP response element modulator), which encodes transcription factors that regulate gene expression in β-cells (50).IBMX increased Crem mRNA by 46-fold and 5-fold with 3 mM or 15 mM glucose, respectively.This result does not support a contribution of Crem-encoded proteins to Rdh5 regulation, because unlike CREM, IBMX affected Rdh5 mRNA to the same extent despite the glucose concentration.Exendin-4 had no effect on Crem mRNA regardless of the glucose concentration, excluding GLP-1 effects.Because IBMX increases both cAMP and cGMP, we tested the impact of riociguat, a guanylate cyclase activator (Fig. 8D).Riociguat suppressed Rdh5 transcription only in the presence of low glucose conditions, indicating that cGMP suppresses Rdh5 transcription only during low glucose and confirming cAMP suppresses Rdh5 transcription in the presence of high glucose.Next, we investigated signaling downstream of glucose-ATP-Ca 2+ .During low glucose, the FoxO1 inhibitor AS1842856 reduced Rdh5 expression by 50%, that is, to the same extent as high glucose (Fig. 8E).With high glucose, the FoxO1    8G).These data suggest that CaMK transmits glucose signaling through Akt and Foxo1 to Rdh5.

9cRA induces β-cell autophagy
Autophagy maintains β-cell mass and function (51).RAR induces autophagy by binding to a retinoic acid receptor response element in the Atg7 promoter (52,53).High glucose decreased Atg7 mRNA relative to lower glucose (Fig. 8F).9cRA restored Atg7 mRNA during high glucose to that of low glucose levels.

Discussion
This report establishes existence of 9cRA in multiple tissues at concentrations similar to or greater than atRA.Identification rested on multiple criteria including the following: (1) selective ion monitoring of multiple tissues relying on comigration of endogenous 9cRA with synthetic 9cRA during highresolution LC, accompanied by selection of the molecular ion in Q1 at m/z 301 [M + H + ], followed by verification of a characteristic product ion in Q3 at m/z 205; (2) a full-scan mass spectrum of endogenous 9cRA revealing the typical molecular ion and product ions after isolation by highresolution LC; (3) derivatization and comigration of the endogenous 9cRA derivative with a synthetic 9cRA derivative during high-resolution LC.Each of these procedures alone would have authenticated the analyte as 9cRA.The three together should resolve unmistakably the presence of 9cRA in tissues.This work also resolved why 9cRA has not been observed previously in tissues other than pancreas.Apparently, a combination of ethanol/saline-based homogenization and tissue-specific matrix effects causes 9cRA to isomerize and/or to extract poorly, reducing it below limits of quantification.Methanol precipitates proteins more efficiently than the saline/ethanol mixture.Homogenization in methanol, with removal of precipitated proteins before hexane extraction, diminishes matrix effects and prevents isomerization.
atRA has been accepted as an autacoid with diverse actions, that is, an endogenous retinoid effector of diverse biological processes (54).Debate over the biological significance of atRA was resolved by demonstrating its occurrence in tissues (55) and the discovery of RAR (56,57).9cRA satisfies the criteria that established the significance of atRA, and therefore should experience acceptance as a retinoid-derived autacoid.The many therapeutic actions of 9cRA and rexinoids contribute to this realization (58).Dissimilar tissue-specific distributions of 9cRA and atRA, and their individual responses to fasting/ refeeding cycles, indicates that they contribute independently to retinoid action, with potentially distinct activities.Rdh5 mRNA and net 9-cis-retinal were normalized to cell protein: ***p < 0.005, ##p < 0.0001 versus control vector.

9-cis-retinoic acid occurs as an energy-regulated autacoid
Because of its catalytic activity with 9-cis-retinol, and its relatively high mRNA expression in pancreas, this work examined the possibility that Rdh5 participates in pancreas 9cRA biosynthesis.Rdh5 is better known as one of the 11-cisretinol dehydrogenases important to the visual cycle (59,60).Mutations in Rdh5 cause the disease fundus albipunctatus which retards dark adaptation and results in night blindness (61,62).Rdh5, however, has equivalent catalytic efficiency (kcat/Km) with 9-cis-retinol and has widespread mRNA expression (46).The current data connects Rdh5 with the first and rate-limiting step of 9cRA biosynthesis.This does not imply that Rdh5 represents the only Rdh that participates in 9cRA biosynthesis.Rdh1 and Rdh10, for example, have activity with cis-retinols (63) Moreover, 9-cis-carotenoids occur in nature and undergo metabolism into 9-cis-retinal, thereby providing a source of cis-retinoids (64).This report also confirms the reports that a glucose challenge reduces pancreas 9cRA.In addition to the nongenomic mechanism of decreasing 9cRA, glucose reduces Rdh5 mRNA by inhibiting its transcription.Glucose, therefore, counteracts the biological functions of 9cRA by reducing its concentrations through both transcriptional and posttranscriptional mechanisms.
FoxO1 serves as the nexus between the counter effects of glucose versus 9cRA in β-cells.Insulin in liver phosphorylates Akt/Pkb, which phosphorylates FoxO1.Phosphorylated FoxO1 translocates from the nucleus to undergo cytosolic proteolysis.The absence of nuclear FoxO1 deactivates the RA biosynthetic pathway by reducing transcription of Rdh1, Rdh10, Dhrs9, Raldh1, Raldh3, and Rbp1 (85).The current work shows that insulin does not affect β-cell 9cRA biosynthesis and Rdh5 mRNA.Instead, glucose metabolism activates Akt independently of insulin to suppress FoxO1.FoxO1 has multiple beneficial effects on β-cells, such as reducing oxidative stress, regulating mitochondria-mediated metabolism, promoting autophagy, and reducing DNA damage, all key processes related to glucotoxicity (86).Activation of Rdh5 transcription by FoxO1 indicates that supporting 9cRA biosynthesis involves another mechanism whereby FoxO1 promotes β-cell fitness.
Calléja et al. (87) excluded an RA serving as a universal ligand for RXRα partnered with PPARβ/δ or RARγ in epidermal suprabasal keratinocytes, in which RARγ transcriptionally represses unknown genes.Formation of lamellar granules in these skin cells requires transcriptional activation by liganded PPARβ/δ/RXRα and transcriptional repression by unliganded RARγ/RXRα, with RARγ dominating RXRα.They concluded that an RA binding to both RARγ and RXRα would produce defective lamellar granules by activating RARγ, Clearly, both RARγ and RXRα cannot undergo activation simultaneously in this context, but neither does RXRα activation seem obligatory.Therefore, explanations other than the one proposed emerge as plausible.For example, the authors noted that retinoids do not occur in mouse keratinocytes.This reveals a physiological mechanism to promote lamellar granule formation, Cyp26s have a well-known function to catabolize RAs to protect retinoid-sensitive locations (83).This established mechanism eclipses the nonphysiological interventions that fostered the conclusion.Suppression of RAR by alternative mechanism(s) might also occur.For example, the glucocorticoid receptor represses RA-induced transcription by interacting with the RAR/RXR transcription complex (41).Finally, with respect to a role for 9cRA as a signaling molecule, it has at least 5-fold higher affinity for RAR than RXR (88).This disparity introduces the possibility that 9cRA may prioritize RAR activation, depending on the tissue concentrations of each RA.This report establishes the presence of 9cRA as an endogenous retinoid in multiple tissues, in concentrations similar to or greater than atRA.Tissue concentrations of 9cRA vary with fasting and refeeding independently of atRA.Glucose decreases 9cRA concentrations and biosynthesis in pancreatic β-cells, whereas 9cRA counterbalances glucotoxicity.These data establish 9cRA as a retinoid-derived autacoid that moderates GSIS in pancreas, promotes β-cell vigor, and likely affects energy use in general.

Experimental procedures
All aspects of these experiments were performed under yellow lights to prevent retinoid isomerization.

Pdx1, HNF4α
Figure 9. Glucose and 9cRA exert opposing actions on β-cells.The glucose transporter Glut2 enables glucose uptake by β-cells.Glucokinase (Gck) catalyzes phosphorylation of intracellular glucose, which undergoes glycolysis to generate pyruvate.Acetyl-CoA formed from pyruvate enters the mitochondria to produce ATP.The increase in ATP induces depolarization of the K + channel, which prompts insulin release (( 91)).Increased ATP also stimulates cAMP biosynthesis via adenylate cyclase (Adcy), which induces calmodulin-dependent protein kinase (CaMK).CamK activates Akt, which phosphorylates the transcription factor FoxO1, resulting in its expulsion from the nucleus.Glucose metabolism also results in the repression of the vital autophagy regulator Atg7.FoxO1 induces expression of Rdh5, which catalyzes the first and rate-limiting reaction in 9cRA biosynthesis.The current data and previous reports show that 9cRA exerts diverse actions on β-cells (17,18).9cRA rapidly reduces activities of Glut2 and Gck through nongenomic mechanisms.It represses transcription of Pdx1 and HNF4α.The latter two transcription factors induce transcription of Glut2 and Gck, decreasing glucose metabolism and thereby tempering GSIS.9cRA also induces Atg7, maintaining β-cell autophagy and vigor.Even though Adcy, CaMK, and Akt affect the activities of PKA, mTOR, and GSK3β, the latter do not affect Rdh5 mRNA.9cRA, 9-cis-retinoic acid; GSIS, glucosestimulated insulin secretion.9-cis-retinoic acid occurs as an energy-regulated autacoid hydrochloride (3-NPH⋅HCl) and N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride (EDC⋅HCl) were purchased from Sigma-Aldrich.Standard 9,13-di-cis-RA was purchased from Santa Cruz Biotechnology.

Animal experiments
C57BL/6J (strain# 000664) mice were purchased from The Jackson Laboratory.Upon arrival, mice were fed a purified AIN93G diet (Research Diets, D10012G) containing 4 IU retinyl acetate/g and 7% fat and bred in-house through >3 generations.Blood, liver, pancreas, kidney, testis, epididymal and inguinal white adipose tissues (eWAT and iWAT), brown adipose tissue (BAT), and brain samples were collected from male 89 weeksold-mice, fasted 16 h and compared to those refed 6 h after a 16 h fast.Mice were euthanized in an isoflurane chamber, followed by cervical dislocation.Blood samples, drawn from the vena cava, were allowed to clot on ice and centrifuged 30 min at 12,000g and 4 C. Sera and tissues were snap-frozen in liquid nitrogen and stored at −80 C until assayed.Animal experimental protocols were approved by the University of California Berkeley Animal Care and Use Committee.

Saline/ethanol homogenization
Tissues (50150 mg) or sera (100 μl) were homogenized in ice-cold 0.9% saline using a glass pestle tissue grinder (PYREX, 77245) with a motorized homogenizer (Heidolph, RZR1) at 280 rpm to produce a 10% homogenate.The homogenate (0.5 ml) was transferred to a 23 ml round-bottom glass tube and mixed with 10 μl internal standard (100 nM 3,3-dimethyl-RA in acetonitrile) and 3 ml of 25 mM KOH, prepared in ethanol, by vortexing for 10 s.Ten milliliters hexane was added.The mixture was vortexed 20 s and centrifuged 2 min at 2000g.The upper hexane phase was discarded.One hundred eighty microliters of 4 M HCl were added to the remaining aqueous phase.RAs were extracted with 10 ml hexane by vortexing 20 s.The upper hexane phase was transferred to a new round bottom glass tube and evaporated under a gentle stream of nitrogen in a water bath (Organomation Associates, N-EVAP 112) at 25 to 30 C. The residue was reconstituted in 40 μl acetonitrile with 20 s vortexing, spun for 10 s at 1200g, and transferred to a glass vial insert.One microliter was injected for LC-APCI-MS/MS analysis.

Methanol homogenization
Tissue samples (50150 mg) or sera (100 μl) were placed into a glass pestle tissue grinder with 1.5 ml of ice-cold methanol and 10 μl of the 100 nM internal standard, prepared in acetonitrile.Samples were homogenized using the motorized homogenizer at 280 rpm.The homogenate was transferred to a 15 ml glass conical tube (PYREX, 8060) and centrifuged 2 min at 1200g at room temperature.The supernatant was transferred to a clean glass round-bottom tube and mixed with 100 μl of 750 mM KOH, prepared in methanol, and vortexed 10 s.To remove hydrophobic impurities, 10 ml of hexane was added, vortexed 10 s, and spun 10 s at 1200g.The upper hexane layer was discarded.The bottom methanol phase was mixed with 180 μl 4 M HCl by vortexing 10 s and extracted with 10 ml hexane.The mixture was centrifuged 2 min at 1200g.The upper hexane layer was transferred to a new round-bottom glass tube and evaporated under a gentle stream of nitrogen with heating at 25 to 30 C in a water bath.The residue was reconstituted in 40 μl of acetonitrile with 20 s vortexing, spun 10 s at 1200g, and transferred to a glass vial insert.One microliter was injected for LC-APCI-MS/MS analysis.

RA derivatization with 3-NPH
Standards of RA isomers (30 pmol each) were prepared in 50 μl acetonitrile.Remaining samples after the injection for RA 9-cis-retinoic acid occurs as an energy-regulated autacoid extraction/quantification were pooled, evaporated under a gentle stream of nitrogen, and reconstituted in 50 μl acetonitrile.Twenty millimolar of 3-NPH was prepared in 40 mM HCl and ethanol (v/v 3:1).One hundred millimolar of EDC was prepared in pyridine and acetonitrile (v/v 1:1).In a 1.8 ml amber glass vial, 50 μl of sample was mixed with 100 μl 3-NPH and 75 μl EDC by vortexing 10 s, then incubated 1 h at room temperature.The reaction mixture was transferred to a 23 ml round-bottom glass tube and extracted with 10 ml hexane as described in the revised extraction section.

Figure 1 .
Figure 1.Comparison of RA resolution and isomerization during extraction.A, structures of RA isomers.Standards of RA isomers (100 fmol each) were resolved by either (B), original LC conditions or (C), revised LC conditions.D, atRA and (F), 9cRA standards in saline/ethanol were extracted and resolved by the original LC conditions.E, atRA and (G), 9cRA standards in methanol were extracted and resolved by the revised LC conditions.Analyses were done by LC/MS/MS using multiple reaction monitoring with a triple quadrupole mass spectrometer, which first isolated the molecular ion at m/z 301 [M + H] + in Q1 and then quantified its product ion at m/z 205 in Q3. 9cRA, 9-cis-retinoic acid; atRA, all-trans-retinoic acid; LC, liquid chromatography; LC/MS/MS, LC-tandem mass spectrometry; RA, retinoic acid.

Figure 2 .
Figure2.Recovery of 9cRA from pancreas without isomerization.A, one microliter of a 100 nM solution of 9cRA in methanol (100 fmol) was injected into LC/MS/MS.B, a pancreas from a male mouse was homogenized in 1.5 ml methanol.Half of the homogenate was analyzed using the revised protocol.C, ten microliters of a 100 nM solution of 9cRA was added to the remaining half of the pancreas homogenate and analyzed using the revised protocol.Recovery was 96%.9cRA, 9-cis-retinoic acid; LC/MS/MS, LC-tandem mass spectrometry.

Figure 3 .
Figure 3. Mass spectrum of endogenous 9cRA.Pancreata from 46 mice were pooled, homogenized in methanol, and applied to LC.The 11.5 to 12.5 min fractions were collected, concentrated, and reinjected for a Q3 product ion scan.A, total ion chromatogram of the reinjected sample.B, Q3 mass scan of endogenous 9cRA.9cRA, 9-cis-retinoic acid; LC, liquid chromatography.

Table 1
Variations of 9cRA and atRA with fasting versus refeeding Mice were fasted 16 h or fasted 16 h and refed 6 h (n = 6, means ± SE). p values refer to the differences between fasted and refed values.Serum 9cRA samples were lost.